Hexuronate c4-epimerase variant having improved d-tagatose conversion activity, and d-tagatose production method using same

ABSTRACT

Provided are a hexuronate C4-epimerase variant with improved activity in converting D-fructose by D-tagatose of hexuronate C4-epimerase and a method for production of D-tagatose using them.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present specification is a divisional of U.S. patent applicationSer. No. 16/060,018, filed Jun. 6, 2018, which is a U.S. National Stageof International Patent Application No. PCT/KR2017/008241 filed Jul. 31,2017, which claims priority to and the benefit of Korean PatentApplication No. 10-2016-0097500 filed in the Korean IntellectualProperty Office on Jul. 29, 2016, the entire contents of which areincorporated herein by reference.

TECHNICAL FIELD

The present invention relates to a hexuronate C4-epimerase variant withimproved conversion activity to D-tagatose and a method for productionof D-tagatose using them.

BACKGROUND ART

Tagatose is a natural sweetener that is present in small amounts infoods such as milk, cheese, cacao, etc., and sweet and natural fruitssuch as apples and tangerines, and also has physical properties similarto those of sugar. Tagatose has a calorie of 1.5 kcal/g, which is aboutone-third of sugar, and has a glycemic index (GI) of 3, which is about5% of the sugar. Tagatose has a variety of health functionalities withsweetness similar to sugar, and thus, tagatose may be used as asubstitute sweetener capable of satisfying health and taste at the sametime when applied to various products.

Conventionally known methods for production of tagatose include achemical (catalytic reaction) method and a biological (isomerizationenzyme reaction) method using galactose as a main raw material (seeKorean Patent Laid-Open Publication No. 2009-0082774). However, lactose,which is a basic raw material of galactose in the above-describedproduction method, is unstable in price due to a production amount,demand and a supply amount of raw milk and lactose in the internationalmarket, and thus there is a limit to stably meet supply and demand ofraw materials for production of tagatose. Therefore, a new method inwhich tagatose is able to be produced by using common generic sugars(sugar, glucose, fructose, etc.) as raw materials has been demanded.Accordingly, the present inventors have reported a production method fortagatose from fructose using a novel hexuronate C4-epimerase (KoreanPatent Laid-Open Publication No. 10-2014-0143109). However, forindustrial production, it is necessary to develop an enzyme havinghigher conversion activity to tagatose.

Under these circumstances, the present inventors confirmed that when anamino acid at a specific position of the hexuronate C4-epimerase wasmutated, the conversion activity from fructose to tagatose wasremarkably increased as compared to that of the wild-type, and completedthe present invention.

Throughout the present specification, a number of patents and documentsare referenced and the citation is shown in parentheses. The disclosuresof these patents and publications are hereby incorporated by referencein their entirety to more clearly illustrate the present invention andthe level of the technical field in which the invention pertains.

DISCLOSURE

The Sequence Listing created on Jun. 6, 2018 with a file size of 5 KB,and filed herewith in ASCII text file format as the file entitled“4207897.TXT,” is hereby incorporated by reference in its entirety.

Technical Problem

An object of the present invention is to provide a hexuronateC4-epimerase variant in which a tyrosine (Y)-403 amino acid residue fromN-terminal of a hexuronate C4-epimerase consisting of an amino acidsequence of SEQ ID NO: 1 is mutated.

Another object of the present invention is to provide a hexuronateC4-epimerase variant in which a threonine (T)-272 amino acid residuefrom N-terminal of a hexuronate C4-epimerase consisting of an amino acidsequence of SEQ ID NO: 1 is mutated.

Still another object of the present invention is to provide a hexuronateC4-epimerase variant in which a serine (S)-185 amino acid residue fromN-terminal of a hexuronate C4-epimerase consisting of an amino acidsequence of SEQ ID NO: 1 is mutated.

Still another object of the present invention is to provide a hexuronateC4-epimerase variant in which a leucine (L)-77 amino acid residue fromN-terminal of a hexuronate C4-epimerase consisting of an amino acidsequence of SEQ ID NO: 1 is mutated.

Still another object of the present invention is to provide a hexuronateC4-epimerase variant in which an alanine (A)-158 amino acid residue fromN-terminal of a hexuronate C4-epimerase consisting of an amino acidsequence of SEQ ID NO: 1 is mutated.

Still another object of the present invention is to provide a hexuronateC4-epimerase variant in which a proline (P)-351 amino acid residue fromN-terminal of a hexuronate C4-epimerase consisting of an amino acidsequence of SEQ ID NO: 1 is mutated.

Still another object of the present invention is to provide a hexuronateC4-epimerase variant in which serine (S)-125, lysine (K)-164, asparticacid (D)-168, and glutamic acid (E)-175 amino acid residues fromN-terminal of a hexuronate C4-epimerase consisting of an amino acidsequence of SEQ ID NO: 1 are mutated.

Still another object of the present invention is to provide a hexuronateC4-epimerase variant in which serine (S)-125, glutamine (Q)-149, andvaline (V)-267 amino acid residues from N-terminal of a hexuronateC4-epimerase consisting of an amino acid sequence of SEQ ID NO: 1 aremutated.

Still another object of the present invention is to provide a nucleicacid encoding a hexuronate C4-epimerase variant disclosed herein, atransformant including the nucleic acid, a microorganism expressing thevariant of the present invention or a culture thereof, or a compositionfor producing D-tagatose comprising the hexuronate C4-epimerase variantof the present invention.

Still another object of the present invention is to provide a method forproduction of D-tagatose comprising contacting the hexuronateC4-epimerase variant of the present invention, a transformant of thepresent invention, a microorganism expressing the variant of the presentinvention or a culture thereof, or a composition for producing tagatoseof the present invention, with D-fructose.

Hereinafter, the present invention is described in more detail. Otherobjects and advantages of the present invention are more apparent fromthe following detailed description together with the appended claims.Descriptions that are not described in the specification can besufficiently recognized and deduced by a person skilled in the technicalfield or fields similar to this, details thereof are omitted.

Technical Solution

In order to accomplish the object of the present invention, according toan exemplary embodiment of the present invention, there is provided ahexuronate C4-epimerase variant in which one or more amino acid residuesselected from the group consisting of histidine (H)-9, tyrosine (Y)-21,glutamic acid (E)-60, valine (V)-62, glutamic acid (E)-68, leucine(L)-77, leucine (L)-91, threonine (T)-97, serine (S)-125, valine(V)-126, leucine (L)-140, aspartic acid (D)-141, tryptophan (W)-145,glutamine (Q)-149, glycine (G)-157, alanine (A)-158, alanine (A)-160,valine (V)-163, lysine (K)-164, proline (P)-166, glutamic acid (E)-167,aspartic acid (D)-168, glutamic acid (E)-175, glycine (G)-176,phenylalanine (F)-177, serine (5)-185, methionine (M)-202, glycine(G)-218, tyrosine (Y)-221, aspartic acid (D)-231, valine (V)-241,tyrosine (Y)-242, valine (V)-267, serine (S)-268, threonine (T)-272,threonine (T)-276, valine (V)-284, phenylalanine (F)-295, phenylalanine(F)-297, phenylalanine (F)-302, tryptophan (W)-306, leucine (L)-316,lysine (K)-337, proline (P)-351, phenylalanine (F)-361, alanine (A)-366,arginine (R)-386, isoleucine (I)-388, serine (S)-402, tyrosine (Y)-403,valine (V)-415, aspartic acid (D)-429, tyrosine (Y)-440, and glycine(G)-441 from N-terminal of hexuronate C4-epimerase consisting of anamino acid sequence of SEQ ID NO: 1 are mutated to other amino acidresidues [see Tables 2 to 10].

According to another exemplary embodiment of the present invention,there is provided a hexuronate C4-epimerase variant in which a tyrosine(Y)-403 amino acid residue from N-terminal of a hexuronate C4-epimeraseconsisting of an amino acid sequence of SEQ ID NO: 1 is mutated.

The tyrosine (Y)-403 amino acid residue may be substituted with alanine(A), cysteine (C), aspartic acid (D), glutamic acid (E), phenylalanine(F), glycine (G), histidine (H), isoleucine (I), lysine (K), leucine(L), methionine (M), asparagine (N), proline (P), glutamine (Q),arginine (R), serine (S), threonine (T), valine (V) or tryptophan (W),and more specifically, may be substituted with phenylalanine (F), serine(S), threonine (T), glutamine (Q), valine (V), alanine (A), orisoleucine (I).

In an exemplary embodiment, in the hexuronate C4-epimerase variant ofthe present invention, a serine (S)-125 amino acid residue from theN-terminal of the hexuronate C4-epimerase consisting of an amino acidsequence of SEQ ID NO: 1 may additionally be mutated in addition to theposition No. 403. The serine (S)-125 amino acid residue may besubstituted with aspartic acid (D), glutamine (Q), glutamic acid (E),threonine (T), asparagine (N), cysteine (C), or tyrosine (Y). In anexample of the exemplary embodiment, the hexuronate C4-epimerase variantof the present invention may be a variant in which the tyrosine (Y)-403amino acid residue is substituted with phenylalanine (F), serine (S),threonine (T), glutamine (Q), or valine (V), and the serine (S)-125amino acid residue is substituted with aspartic acid (D).

In an example of the exemplary embodiment, the hexuronate C4-epimerasevariant of the present invention may be a variant in which one or moreamino acid residues selected from the group consisting of serine(S)-185, valine (V)-267, serine (S)-268, threonine (T)-272, tryptophan(W)-306, and arginine (R)-386 from the N-terminal of the hexuronateC4-epimerase consisting of the amino acid sequence of SEQ ID NO: 1 mayadditionally be mutated in addition to the tyrosine (Y)-403 amino acidresidue and the serine (S)-125 amino acid residue. The serine (S)-185may be substituted with lysine (K), arginine (R), histidine (H),glutamine (Q), alanine (A) or glycine (G); the valine (V)-267 may besubstituted with methionine (M); the serine (S)-268 may be substitutedwith cysteine (C) or threonine (T); the threonine (T)-272 may besubstituted with alanine (A), aspartic acid (D), glutamic acid (E),phenylalanine (F), glycine (G), histidine (H), isoleucine (I), lysine(K), leucine (L), methionine (M), asparagine (N), proline (P), glutamine(Q), arginine (R), serine (S), valine (V) or tyrosine (Y); thetryptophan (W)-306 may be substituted with phenylalanine (F), histidine(H), methionine (M) or valine (V); the arginine (R)-386 may besubstituted with proline (P) or valine (V).

In an example of the exemplary embodiment, the present invention mayprovide a variant which is mutated at any one position of the serine(S)-185, the valine (V)-267, the serine (S)-268, the threonine (T)-272,the tryptophan (W)-306, and the arginine (R)-386, in addition to themutations of the tyrosine (Y)-403 amino acid residue and the serine(S)-125 amino acid residue. For example, the hexuronate C4-epimerasevariant of the present invention may be a variant in which the tyrosine(Y)-403 amino acid residue is substituted with phenylalanine (F), serine(S), threonine (T), glutamine (Q), valine (V), alanine (A), orisoleucine (I), the serine (S)-125 amino acid residue is substitutedwith aspartic acid (D), and the serine (S)-185 amino acid residue issubstituted with lysine (K), histidine (H), or glutamine (Q). In anexample, the hexuronate C4-epimerase variant of the present inventionmay be a variant in which the tyrosine (Y)-403 amino acid residue issubstituted with phenylalanine (F), serine (S), threonine (T), glutamine(Q), valine (V), alanine (A), or isoleucine (I), the serine (S)-125amino acid residue is substituted with aspartic acid (D), and the valine(V)-267 amino acid residue is substituted with methionine (M). In anexample, the hexuronate C4-epimerase variant of the present inventionmay be a variant in which the tyrosine (Y)-403 amino acid residue issubstituted with phenylalanine (F), serine (S), threonine (T), glutamine(Q), valine (V), alanine (A), or isoleucine (I), the serine (S)-125amino acid residue is substituted with aspartic acid (D), and the serine(S)-268 amino acid residue is substituted with cysteine (C) or threonine(T). In an example, the hexuronate C4-epimerase variant of the presentinvention may be a variant in which the tyrosine (Y)-403 amino acidresidue is substituted with phenylalanine (F), serine (S), threonine(T), glutamine (Q), valine (V), alanine (A), or isoleucine (I), theserine (S)-125 amino acid residue is substituted with aspartic acid (D),and the threonine (T)-272 amino acid residue is substituted withaspartic acid (D). In an example, the hexuronate C4-epimerase variant ofthe present invention may be a variant in which the tyrosine (Y)-403amino acid residue is substituted with phenylalanine (F), serine (S),threonine (T), glutamine (Q), valine (V), alanine (A), or isoleucine(I), the serine (S)-125 amino acid residue is substituted with asparticacid (D), and the tryptophan (W)-306 amino acid residue is substitutedwith phenylalanine (F), histidine (H), methionine (M) or valine (V). Inan example, the hexuronate C4-epimerase variant of the present inventionmay be a variant in which the tyrosine (Y)-403 amino acid residue issubstituted with phenylalanine (F), serine (S), threonine (T), glutamine(Q), valine (V), alanine (A), or isoleucine (I), the serine (S)-125amino acid residue is substituted with aspartic acid (D), and thearginine (R)-386 amino acid residue is substituted with proline (P) orvaline (V).

In another example of the exemplary embodiment, the hexuronateC4-epimerase variant of the present invention may be a variant which issubstituted at any two positions selected from the serine (S)-185, thevaline (V)-267, the serine (S)-268, the threonine (T)-272, thetryptophan (W)-306, and the arginine (R)-386, in addition to thetyrosine (Y)-403 amino acid residue and the serine (S)-125 amino acidresidue. The mutation position may be positions at 185 and 267,positions at 185 and 268, positions at 185 and 272, positions at 185 and306, positions at 185 and 386, positions at 267 and 268, positions at267 and 272, positions at 267 and 306, positions at 267 and 386,positions at 268 and 272, positions at 268 and 306, positions at 268 and386, positions at 272 and 306, positions at 272 and 386, or positions at306 and 386. For example, the hexuronate C4-epimerase variant of thepresent invention may be a variant in which the tyrosine (Y)-403 aminoacid residue is substituted with phenylalanine (F), serine (S),threonine (T), glutamine (Q), valine (V), alanine (A), or isoleucine(I), the serine (S)-125 amino acid residue is substituted with asparticacid (D), the serine (S)-185 amino acid residue is substituted withlysine (K), histidine (H), or glutamine (Q), and the valine (V)-267 issubstituted with methionine (M). In an example, the hexuronateC4-epimerase variant of the present invention may be a variant in whichthe tyrosine (Y)-403 amino acid residue is substituted withphenylalanine (F), serine (S), threonine (T), glutamine (Q), valine (V),alanine (A), or isoleucine (I), the serine (S)-125 amino acid residue issubstituted with aspartic acid (D), the serine (S)-185 amino acidresidue is substituted with lysine (K), histidine (H), or glutamine (Q),and the serine (S)-268 amino acid residue is substituted with cysteine(C) or threonine (T). In an example, the hexuronate C4-epimerase variantof the present invention may be a variant in which the tyrosine (Y)-403amino acid residue is substituted with phenylalanine (F), serine (S),threonine (T), glutamine (Q), valine (V), alanine (A), or isoleucine(I), the serine (S)-125 amino acid residue is substituted with asparticacid (D), the serine (S)-185 amino acid residue is substituted withlysine (K), histidine (H), or glutamine (Q), and the threonine (T)-272is substituted with aspartic acid (D). In an example, the hexuronateC4-epimerase variant of the present invention may be a variant in whichthe tyrosine (Y)-403 amino acid residue is substituted withphenylalanine (F), serine (S), threonine (T), glutamine (Q), valine (V),alanine (A), or isoleucine (I), the serine (S)-125 amino acid residue issubstituted with aspartic acid (D), the serine (S)-268 amino acidresidue is substituted with cysteine (C) or threonine (T), and thetryptophan (W)-306 is substituted with phenylalanine (F), histidine (H),methionine (M) or valine (V). In an example, the hexuronate C4-epimerasevariant of the present invention may be a variant in which the tyrosine(Y)-403 amino acid residue is substituted with phenylalanine (F), serine(S), threonine (T), glutamine (Q), valine (V), alanine (A), orisoleucine (I), the serine (S)-125 amino acid residue is substitutedwith aspartic acid (D), the serine (S)-268 amino acid residue issubstituted with cysteine (C) or threonine (T), and the arginine (R)-386amino acid residue is substituted with proline (P) or valine (V).

In another example of the exemplary embodiment, the hexuronateC4-epimerase variant of the present invention may be a variant which issubstituted at any three positions selected from the serine (S)-185, thevaline (V)-267, the threonine (T)-272, the tryptophan (W)-306, and thearginine (R)-386, in addition to the tyrosine (Y)-403 amino acid residueand the serine (S)-125 amino acid residue. The mutation position may be,for example, the positions at 185, 267, and 268; the positions at 185,267, and 272; the positions at 185, 267, and 306; the positions at 185,267, and 386; the positions at 185, 268, and 272; the positions at 185,268, and 306; the positions at 185, 268, and 386; the positions at 185,272, and 306; the positions at 185, 272, and 386; the positions at 267,268, and 272; the positions at 267, 268, and 306; the positions at 267,268, and 386; the positions at 267, 272, and 306; the positions at 267,272, and 386; the positions at 267, 386, and 306; the positions at 268,272, and 306; the positions at 268, 272, and 386; the positions at 268,306, and 386; or the positions at 272, 306, and 386. In an example, thehexuronate C4-epimerase variant of the present invention may be avariant in which the tyrosine (Y)-403 amino acid residue is substitutedwith phenylalanine (F), serine (S), threonine (T), glutamine (Q), valine(V), alanine (A), or isoleucine (I), the serine (S)-125 amino acidresidue is substituted with aspartic acid (D), the serine (S)-185 aminoacid residue is substituted with lysine (K), histidine (H), or glutamine(Q), the valine (V)-267 amino acid residue is substituted withmethionine (M), and the serine (S)-268 amino acid residue is substitutedwith cysteine (C) or threonine (T). For example, the hexuronateC4-epimerase variant of the present invention may be a variant in whichthe tyrosine (Y)-403 amino acid residue is substituted withphenylalanine (F), serine (S), threonine (T), glutamine (Q), valine (V),alanine (A), or isoleucine (I), the serine (S)-125 amino acid residue issubstituted with aspartic acid (D), the serine (S)-185 amino acidresidue is substituted with lysine (K), histidine (H), or glutamine (Q),the valine (V)-267 amino acid residue is substituted with methionine(M), and the threonine (T)-272 is substituted with aspartic acid (D).For example, the hexuronate C4-epimerase variant of the presentinvention may be a variant in which the tyrosine (Y)-403 amino acidresidue is substituted with phenylalanine (F), serine (S), threonine(T), glutamine (Q), valine (V), alanine (A), or isoleucine (I), theserine (S)-125 amino acid residue is substituted with aspartic acid (D),the serine (S)-185 amino acid residue is substituted with lysine (K),histidine (H), or glutamine (Q), the valine (V)-267 amino acid residueis substituted with methionine (M), and the tryptophan (W)-306 aminoacid residue is substituted with phenylalanine (F), histidine (H),methionine (M) or valine (V).

In another example of the exemplary embodiment, the hexuronateC4-epimerase variant of the present invention may be a variant which issubstituted at any four, five or six positions selected from the serine(S)-185, the valine (V)-267, the serine (S)-268, the threonine (T)-272,the tryptophan (W)-306, and the arginine (R)-386, in addition to thetyrosine (Y)-403 amino acid residue and the serine (5)-125 amino acidresidue.

In an exemplary embodiment, in the hexuronate C4-epimerase variant ofthe present invention, the lysine (K)-164, the aspartic acid (D)-168,the glutamic acid (E)-175, the asparagine (N)-297, and the isoleucine(I)-388 from the N-terminal of the hexuronate C4-epimerase consisting ofthe amino acid sequence of SEQ ID NO: 1 may additionally be mutated inaddition to the tyrosine (Y)-403, serine (S)-125, and serine (S)-268amino acid residues. The lysine (K)-164 may be substituted withmethionine (M), the aspartic acid (D)-168 may be substituted withglutamic acid (E), the glutamic acid (E)-175 may be substituted withglycine (G), the asparagine (N)-297 may be substituted with lysine (K),and the isoleucine (I)-388 may be substituted with valine (V). Thehexuronate C4-epimerase variant of an example of the exemplaryembodiment may be a variant in which the tyrosine (Y)-403 amino acidresidue is substituted with phenylalanine (F), serine (S), threonine(T), glutamine (Q), valine (V), alanine (A), or isoleucine (I), theserine (5)-125 amino acid residue is substituted with aspartic acid (D),the serine (S)-268 amino acid residue is substituted with cysteine (C)or threonine (T), and the lysine (K)-164 is substituted with methionine(M), the aspartic acid (D)-168 is substituted with glutamic acid (E),the glutamic acid (E)-175 is substituted with glycine (G), theasparagine (N)-297 is substituted with lysine (K), and the isoleucine(I)-388 is substituted with valine (V).

In an exemplary embodiment, in the hexuronate C4-epimerase variant ofthe present invention, the proline (P)-351 from the N-terminal of thehexuronate C4-epimerase consisting of the amino acid sequence of SEQ IDNO: 1 may additionally be mutated in addition to the tyrosine (Y)-403,serine (S)-125, valine (V)-267, and arginine (R)-386 amino acidresidues. Specifically, the proline (P)-351 may be substituted withserine (S).

In an exemplary embodiment, in the hexuronate C4-epimerase variant ofthe present invention, the glutamic acid (E)-68 from the N-terminal ofthe hexuronate C4-epimerase consisting of the amino acid sequence of SEQID NO: 1 may additionally be mutated in addition to the tyrosine(Y)-403, serine (S)-125, serine (S)-185, valine (V)-267, and tryptophan(W)-306 amino acid residues. The glutamic acid (E)-68 may be substitutedwith glycine (G).

In an exemplary embodiment, in the hexuronate C4-epimerase variant ofthe present invention, the glutamic acid (E)-60, the methionine (M)-202,the tyrosine (Y)-221 and the tyrosine (Y)-242 from the N-terminal of thehexuronate C4-epimerase consisting of the amino acid sequence of SEQ IDNO: 1 may additionally be mutated in addition to the tyrosine (Y)-403,serine (S)-125, valine (V)-267, serine (S)-268, and arginine (R)-386amino acid residues. The glutamic acid (E)-60 may be substituted withaspartic acid (D), the methionine (M)-202 may be substituted withthreonine (T), the tyrosine (Y)-221 may be substituted withphenylalanine (F), and the tyrosine (Y)-242 may be substituted withphenylalanine (F).

In an exemplary embodiment, in the hexuronate C4-epimerase variant ofthe present invention, one or more amino acid residues selected from thegroup consisting of the serine (S)-185, the valine (V)-267, the serine(S)-268, the threonine (V)-272, the tryptophan (W)-306, and the arginine(R)-386 may additionally be mutated in addition to the tyrosine (Y)-403and serine (S)-125 amino acid residues, and in the hexuronateC4-epimerase variant of the present invention, one or more amino acidresidues selected from the group consisting of the leucine (L)-91, theaspartic acid (D)-141 and the glycine (G)-176 from the N-terminal of thehexuronate C4-epimerase consisting of the amino acid sequence of SEQ IDNO: 1 may additionally be mutated. Specifically, in the hexuronateC4-epimerase variant of the present invention, the leucine (L)-91, theaspartic acid (D)-141 or the glycine (G)-176 amino acid residue mayadditionally be mutated in addition to the tyrosine (Y)-403, serine(S)-125, serine (S)-185, valine (V)-267, serine (5)-268 and threonine(T)-272 amino acid residues. The leucine (L)-91 may be substituted withtryptophan (W), isoleucine (I), or asparagine (N), the aspartic acid(D)-141 may be substituted with phenylalanine (F), and the glycine(G)-176 may be substituted with histidine (H), phenylalanine (F) ortyrosine (Y).

In an exemplary embodiment, in the hexuronate C4-epimerase variant ofthe present invention, one or more amino acid residues selected from thegroup consisting of the valine (V)-267, the serine (S)-268, thethreonine (T)-272, and the tryptophan (W)-306 may additionally bemutated in addition to the tyrosine (Y)-403 and the serine (S)-125 aminoacid residues, and in the hexuronate C4-epimerase variant, the valine(V)-284 or the valine (V)-415 from the N-terminal of the hexuronateC4-epimerase consisting of the amino acid sequence of SEQ ID NO: 1 mayadditionally be mutated. Specifically, in the hexuronate C4-epimerasevariant of the present invention, the valine (V)-284 and valine (V)-415amino acid residues may additionally be mutated in addition to thetyrosine (Y)-403, serine (S)-125, valine (V)-267, serine (S)-268,threonine (T)-272, and tryptophan (W)-306 amino acid residues. Thevaline (V)-284 may be substituted with alanine (A), and the valine(V)-415 may be substituted with glutamic acid (E).

In an exemplary embodiment, in the hexuronate C4-epimerase variant ofthe present invention, one or more amino acid residues selected from thegroup consisting of the serine (S)-185, the valine (V)-267, the serine(S)-268, the threonine (V)-272, and the tryptophan (W)-306 mayadditionally be mutated in addition to the tyrosine (Y)-403 and serine(S)-125 amino acid residues, and in the hexuronate C4-epimerase variant,the proline (P)-166 or the aspartic acid (D)-231 from the N-terminal ofthe hexuronate C4-epimerase consisting of the amino acid sequence of SEQID NO: 1 may additionally be mutated. Specifically, in the hexuronateC4-epimerase variant of the present invention, the proline (P)-166 orthe aspartic acid (D)-231 may additionally be mutated in addition to thetyrosine (Y)-403, serine (S)-125, serine (S)-185, valine (V)-267, serine(5)-268, threonine (T)-272, and tryptophan (W)-306 amino acid residues.The proline (P)-166 may be substituted with arginine (R), and theaspartic acid (D)-231 may be substituted with arginine (R).

In an exemplary embodiment, in the hexuronate C4-epimerase variant ofthe present invention, the serine (5)-185, valine (V)-267, serine(S)-268, threonine (V)-272, the tryptophan (W) residue at position 306and the arginine (R) residue at position 386 may additionally be mutatedin addition to the tyrosine (Y)-403 and serine (S)-125 amino acidresidues, and in the hexuronate C4-epimerase variant, the valine (V)-126from the N-terminal of the hexuronate C4-epimerase consisting of theamino acid sequence of SEQ ID NO: 1 may additionally be mutated.Specifically, in the hexuronate C4-epimerase variant of the presentinvention, the valine (V)-126 may additionally be mutated in addition tothe tyrosine (Y)-403, serine (S)-125, serine (S)-185, valine (V)-267,serine (S)-268, threonine (T)-272, and tryptophan (W)-386 amino acidresidues. The valine (V)-126 may be substituted with alanine (A),phenylalanine (F), glycine (G), isoleucine (I), leucine (L), proline(P), asparagine (R) or threonine (T).

According to an exemplary embodiment of the present invention, thehexuronate C4-epimerase variant of the present invention may be ahexuronate C4-epimerase variant in which the tyrosine (Y)-403, serine(S)-125, serine (5)-185, valine (V)-267, serine (S)-268, threonine(T)-272, tryptophan (W)-306, and arginine (R)-386 amino acid residuesfrom the N-terminal of the hexuronate C4-epimerase consisting of theamino acid sequence of SEQ ID NO: 1 are mutated.

In an exemplary embodiment, in the hexuronate C4-epimerase variant ofthe present invention, the threonine (T)-97, the valine (V)-126, thetryptophan (W)-145, the valine (V)-163, the lysine (K)-164, the proline(P)-166, the aspartic acid (D)-231, the valine (V)-241, the threonine(T)-276, the lysine (K)-337, the alanine (A)-366, the serine (S)-402,the aspartic acid (D)-429 or the tyrosine (Y)-440 amino acid residue mayadditionally be mutated in addition to the tyrosine (Y)-403, serine(S)-125, serine (S)-185, valine (V)-267, serine (S)-268, threonine(T)-272, and arginine (R)-386 amino acid residues of SEQ ID NO: 1. Thethreonine (T)-97 may be substituted with alanine (A) or leucine (L); thevaline (V)-126 may be substituted with phenylalanine (F), leucine (L),proline (P), isoleucine (I), threonine (T), alanine (A), glycine (G) orarginine (R); the tryptophan (W)-145 may be substituted with alanine(A); the valine (V)-163 may be substituted with alanine (A), methionine(M) or glutamine (Q); the lysine (K) −164 may be substituted withmethionine (M); the proline (P)-166 may be substituted with arginine(R); the aspartic acid (D)-231 may be substituted with arginine (R); thevaline (V)-241 may be substituted with asparagine (N), threonine (T) orserine (S); the threonine (T)-276 may be substituted with glutamic acid(E) or alanine (A); the lysine (K)-337 may be substituted with glutamicacid (E), phenylalanine (F), asparagine (N), proline (P), serine (S),threonine (T), tryptophan (W) or tyrosine (Y); the alanine (A)-366 maybe substituted with serine (S), glycine (G) or cysteine (C); the serine(S)-402 may be substituted with phenylalanine (F), cysteine (C) ortyrosine (Y); the aspartic acid (D)-429 may be substituted with proline(P), and the tyrosine (Y)-440 may be substituted with alanine (A).

In an exemplary embodiment, in the hexuronate C4-epimerase variant ofthe present invention, the lysine (K)-164, the aspartic acid (D)-166 orthe aspartic acid (D)-231 amino acid residue from the N-terminal of thehexuronate C4-epimerase consisting of the amino acid sequence of SEQ IDNO: 1 may additionally be mutated in addition to the tyrosine (Y)-403,serine (S)-125, serine (S)-185, valine (V)-267, serine (S)-268,threonine (T)-272, arginine (R) −386, and threonine (T)-97 amino acidresidues. The lysine (K)-164 may be substituted with methionine (M); theaspartic acid (D)-166 may be substituted with arginine (R); and theaspartic acid (D)-231 may be substituted with arginine (R).

In an exemplary embodiment, in the hexuronate C4-epimerase variant ofthe present invention, the aspartic acid (D)-231 amino acid residue fromthe N-terminal of the hexuronate C4-epimerase consisting of the aminoacid sequence of SEQ ID NO: 1 may additionally be mutated in addition tothe tyrosine (Y)-403, serine (S)-125, serine (S)-185, valine (V)-267,serine (S)-268, threonine (T)-272, arginine (R)-386, and valine (V)-163amino acid residues. The aspartic acid (D)-231 may be substituted witharginine (R).

In an exemplary embodiment, in the hexuronate C4-epimerase variant ofthe present invention, the glycine (G)-157, the alanine (A)-160, theglutamic acid (E)-167, the phenylalanine (F)-177, the glycine (G)-218,the phenylalanine (F)-295, the phenylalanine (F)-302, the phenylalanine(F)-361, the alanine (A)-366 or the glycine (G)-441 amino acid residuefrom the N-terminal of the hexuronate C4-epimerase consisting of theamino acid sequence of SEQ ID NO: 1 may additionally be mutated inaddition to the tyrosine (Y)-403, serine (S)-125, serine (S)-185, valine(V)-267, serine (S)-268, threonine (T)-272, arginine (R)-386, and lysine(K)-337 amino acid residues. The glycine (G)-157 may be substituted witharginine (R); the alanine (A)-160 may be substituted with leucine (L),phenylalanine (F), arginine (R) or tyrosine (Y); the glutamic acid(E)-167 may be substituted with alanine (A), tryptophan (W), isoleucine(I), lysine (K), methionine (M), valine (V) or serine (S); thephenylalanine (F)-177 may be substituted with tyrosine (Y), histidine(H) or leucine (L); the glycine (G)-218 may be substituted withisoleucine (I), serine (S), leucine (L), phenylalanine (F) or cysteine(C); the phenylalanine (F)-295 may be substituted with cysteine (C),arginine (R) or tyrosine (Y); the phenylalanine (F)-302 may besubstituted with cysteine (C); the phenylalanine (F)-361 may besubstituted with lysine (K), glutamic acid (E), valine (V), tryptophan(W), tyrosine (Y), methionine (M), arginine (R), glutamine (Q), leucine(L) or cysteine (C); the alanine (A)-366 may be substituted with serine(S); the glycine (G)-441 may be substituted with glutamic acid (E),tryptophan (W), histidine (H), lysine (K), alanine (A), arginine (R),serine (S) or phenylalanine (F).

In an exemplary embodiment, in the hexuronate C4-epimerase variant ofthe present invention, the leucine (L)-77 amino acid residue, thealanine (A) −158 amino acid residue, or a combination of the amino acidresidues from the N-terminal of the hexuronate C4-epimerase consistingof the amino acid sequence of SEQ ID NO: 1 may additionally be mutatedin addition to the tyrosine (Y)-403 amino acid residue and the serine(S)-125 amino acid residue. The leucine (L)-77 may be substituted withproline (P) or arginine (R), and the alanine (A)-158 may be substitutedwith threonine (T). In the hexuronate C4-epimerase variant in which thetyrosine (Y)-403, the serine (S)-125, the leucine (L) −77 amino acidresidue, and the alanine (A) −158 amino acid residue are mutated, thearginine (R)-386 amino acid residue from the N-terminal of thehexuronate C4-epimerase consisting of the amino acid sequence of SEQ IDNO: 1 may additionally be mutated. The arginine (R)-386 may besubstituted with proline (P) or valine (V).

According to another exemplary embodiment of the present invention,there is provided a hexuronate C4-epimerase variant in which a serine(S)-185 amino acid residue from N-terminal of a hexuronate C4-epimeraseconsisting of an amino acid sequence of SEQ ID NO: 1 is mutated. Theserine (S)-185 amino acid residue may be substituted with alanine (A),glycine (G), histidine (H), lysine (K), glutamine (Q), or arginine (R).

In an exemplary embodiment, in the hexuronate C4-epimerase variant ofthe present invention, the serine (5)-125 amino acid residue mayadditionally be mutated in addition to the position No. 185. The serine(S)-125 amino acid residue may be substituted with cysteine (C),tyrosine (Y), glutamine (Q), glutamic acid (E), threonine (T),asparagine (N), or aspartic acid (D). In an example of the exemplaryembodiment, the hexuronate C4-epimerase variant of the present inventionmay be a variant in which the serine (S)-185 amino acid residue issubstituted with alanine (A), glycine (G), histidine (H), lysine (K),glutamine (Q), or arginine (R), and the serine (S)-125 amino acidresidue is substituted with cysteine (C), tyrosine (Y), glutamine (Q),glutamic acid (E), threonine (T), asparagine (N), or aspartic acid (D).In a specific example, the hexuronate C4-epimerase variant of thepresent invention may be a variant in which the serine (S)-185 aminoacid residue is substituted with alanine (A), glycine (G), histidine(H), lysine (K), glutamine (Q), or arginine (R), and the serine (S)-125amino acid residue is substituted with aspartic acid (D).

In an exemplary embodiment, in the hexuronate C4-epimerase variant ofthe present invention, the serine (5)-268 amino acid residue from theN-terminal of the hexuronate C4-epimerase consisting of the amino acidsequence of SEQ ID NO: 1 is additionally mutated in addition to theserine (5)-185 and the serine (S)-125. The serine (S)-268 may besubstituted with cysteine (C) or threonine (T).

According to another exemplary embodiment of the present invention,there is provided a hexuronate C4-epimerase variant in which thethreonine (T)-272 amino acid residue from the N-terminal of a hexuronateC4-epimerase consisting of the amino acid sequence of SEQ ID NO: 1 ismutated. The threonine (T)-272 may be substituted with alanine (A),aspartic acid (D), glutamic acid (E), phenylalanine (F), glycine (G),histidine (H), isoleucine (L), lysine (K), leucine (L), methionine (M),asparagine (N), proline (P), glutamine (Q), arginine (R), serine (S),valine (V) or tyrosine (Y).

In an exemplary embodiment, in the hexuronate C4-epimerase variant ofthe present invention, the serine (S)-125 amino acid residue mayadditionally be mutated in addition to the threonine (T)-272 amino acidresidue. The serine (S)-125 amino acid residue may be substituted withcysteine (C), tyrosine (Y), glutamine (Q), glutamic acid (E), threonine(T), asparagine (N), or aspartic acid (D). Accordingly, the hexuronateC4-epimerase variant of the present invention may be a variant in whichthe threonine (T)-272 is substituted with alanine (A), aspartic acid(D), glutamic acid (E), phenylalanine (F), glycine (G), histidine (H),isoleucine (I), lysine (K), leucine (L), methionine (M), asparagine (N),proline (P), glutamine (Q), arginine (R), serine (S), valine (V) ortyrosine (Y), and the serine (S)-125 is substituted with cysteine (C),tyrosine (Y), glutamine (Q), glutamic acid (E), threonine (T),asparagine (N) or aspartic acid (D). In an example, the hexuronateC4-epimerase variant of the present invention may be a variant in whichthe threonine (T)-272 is substituted with serine (S), proline (P),aspartic acid (D), histidine (H), glutamine (Q), asparagine (N), lysine(K) or tyrosine (Y), and the serine (S)-125 is substituted with asparticacid (D).

In an exemplary embodiment, in the hexuronate C4-epimerase variant ofthe present invention, the serine (S)-185 amino acid residue from theN-terminal of the hexuronate C4-epimerase consisting of the amino acidsequence of SEQ ID NO: 1 may additionally be mutated in addition to thethreonine (T)-272 and serine (S)-125 amino acid residues. The serine(S)-185 amino acid residue may be substituted with alanine (A), glycine(G), histidine (H), lysine (K), glutamine (Q), or arginine (R). In anexemplary embodiment, the hexuronate C4-epimerase variant of the presentinvention may be a variant in which the threonine (T)-272 is substitutedwith aspartic acid (D), valine (V), isoleucine (I), leucine (L),methionine (M), glutamine (Q), or serine (S), the serine (S)-125 issubstituted with aspartic acid (D), and the serine (S)-185 issubstituted with lysine (K).

In an example of the exemplary embodiment, in the hexuronateC4-epimerase variant of the present invention, one or more amino acidresidues selected from the group consisting of the valine (V)-267, theserine (S)-268, and the tryptophan (W)-306 from the N-terminal of thehexuronate C4-epimerase consisting of the amino acid sequence of SEQ IDNO: 1 are additionally mutated in addition to the threonine (T)-272,serine (S)-125, and serine (S)-185 amino acid residues. The variant maybe a variant in which the valine (V)-267 is substituted with methionine(M), the serine (5)-268 is substituted with cysteine (C) or threonine(T), and the tryptophan (W)-306 is substituted with phenylalanine (F),histidine (H), methionine (M) or valine (V). In an exemplary embodiment,the hexuronate C4-epimerase variant of the present invention may be avariant in which the threonine (T)-272 is substituted with aspartic acid(D), valine (V), isoleucine (I), leucine (L), methionine (M), glutamine(Q), Or serine (S), the serine (S)-125 is substituted with aspartic acid(D), the serine (S)-185 is substituted with lysine (K), and the valine(V)-267 is substituted with methionine (M). In an exemplary embodiment,the hexuronate C4-epimerase variant of the present invention may be avariant in which the threonine (T)-272 is substituted with aspartic acid(D), valine (V), isoleucine (I), leucine (L), methionine (M), glutamine(Q), or serine (S), the serine (S)-125 is substituted with aspartic acid(D), the serine (S)-185 is substituted with lysine (K), and the serine(S)-268 is substituted with cysteine (C) or threonine (T). In anexemplary embodiment, the hexuronate C4-epimerase variant of the presentinvention may be a variant in which the threonine (T)-272 is substitutedwith aspartic acid (D), valine (V), isoleucine (I), leucine (L),methionine (M), glutamine (Q), or serine (S), the serine (S)-125 issubstituted with aspartic acid (D), the serine (S)-185 is substitutedwith lysine (K), and the tryptophan (W)-306 is substituted withphenylalanine (F), histidine (H), methionine (M) or valine (V). In anexemplary embodiment, the hexuronate C4-epimerase variant of the presentinvention may be a variant in which the threonine (T)-272 is substitutedwith aspartic acid (D), valine (V), isoleucine (I), leucine (L),methionine (M), glutamine (Q), or serine (S), the serine (S)-125 issubstituted with aspartic acid (D), the serine (S)-185 is substitutedwith lysine (K), the valine (V)-267 is substituted with methionine (M),and the serine (S)-268 is substituted with cysteine (C) or threonine(T). In an exemplary embodiment, the hexuronate C4-epimerase variant ofthe present invention may be a variant in which the threonine (T)-272 issubstituted with aspartic acid (D), valine (V), isoleucine (I), leucine(L), methionine (M), glutamine (Q), or serine (S), the serine (S)-125 issubstituted with aspartic acid (D), the serine (S)-185 is substitutedwith lysine (K), the valine (V)-267 is substituted with methionine (M),and the tryptophan (W)-306 is substituted with phenylalanine (F),histidine (H), methionine (M) or valine (V). In an exemplary embodiment,the hexuronate C4-epimerase variant of the present invention may be avariant in which the threonine (T)-272 is substituted with aspartic acid(D), valine (V), isoleucine (I), leucine (L), methionine (M), glutamine(Q), or serine (S), the serine (S)-125 is substituted with aspartic acid(D), the serine (S)-185 is substituted with lysine (K), the serine(S)-268 is substituted with cysteine (C) or threonine (T), and thetryptophan (W)-306 is substituted with phenylalanine (F), histidine (H),methionine (M) or valine (V). In an exemplary embodiment, the hexuronateC4-epimerase variant of the present invention may be a variant in whichthe threonine (T)-272 is substituted with aspartic acid (D), valine (V),isoleucine (I), leucine (L), methionine (M), glutamine (Q), or serine(S), the serine (S)-125 is substituted with aspartic acid (D), theserine (S)-185 is substituted with lysine (K), the valine (V)-267 issubstituted with methionine (M), the serine (S)-268 is substituted withcysteine (C) or threonine (T), and the tryptophan (W)-306 is substitutedwith phenylalanine (F), histidine (H), methionine (M) or valine (V).

In an exemplary embodiment, the hexuronate C4-epimerase variant of thepresent invention may be a variant in which the valine (V)-267 residue,the serine (S)-268 residue, or a combination of the valine (V)-267residue and the serine (S)-268 residue is additionally mutated inaddition to the threonine (T)-272 and serine (S)-125 residues. Thevaline (V)-267 residue may be substituted with methionine (M), and theserine (S)-268 may be substituted with cysteine (C) or threonine (T). Inan exemplary embodiment, the hexuronate C4-epimerase variant of thepresent invention may be a variant in which the threonine (T)-272 issubstituted with aspartic acid (D), valine (V), isoleucine (I), leucine(L), methionine (M), glutamine (Q), or serine (S), the serine (S)-125 issubstituted with aspartic acid (D), the valine (V)-267 is substitutedwith methionine (M), and the serine (S)-268 is substituted with cysteine(C) or threonine (T).

In an exemplary embodiment, the hexuronate C4-epimerase variant of thepresent invention may be a variant in which aspartic acid (D)-231, thearginine (R)-386, or a combination thereof from the N-terminal of thehexuronate C4-epimerase consisting of the amino acid sequence of SEQ IDNO: 1 is additionally mutated in addition to the threonine (T)-272 andthe serine (S)-125; and the valine (V)-267 and/or the serine (S)-268.The aspartic acid (D)-231 may be substituted with arginine (R), and thearginine (R)-386 may be substituted with proline (P) or valine (V). Inan exemplary embodiment, the hexuronate C4-epimerase variant of thepresent invention may be a variant in which the threonine (T)-272 issubstituted with aspartic acid (D), valine (V), isoleucine (I), leucine(L), methionine (M), glutamine (Q), Or serine (S), the serine (S)-125 issubstituted with aspartic acid (D), the valine (V)-267 is substitutedwith methionine (M), the serine (S)-268 is substituted with cysteine(C)or threonine (T), the aspartic acid (D)-231 is substituted with arginine(R), the arginine (R)-386 is substituted with proline (P) or valine (V),or both position Nos. 231 and 386 are substituted with arginine (R), orproline (P) or valine (V), respectively.

In an exemplary embodiment, the hexuronate C4-epimerase variant of thepresent invention may be variant in which one or more amino acidresidues selected from the group consisting of the threonine (T)-97, theglutamine (Q)-149, the proline (P)-166, or the proline (P)-351 from theN-terminal of the hexuronate C4-epimerase consisting of the amino acidsequence of SEQ ID NO: 1 are additionally mutated in addition to thethreonine (T)-272, the serine (S)-125, the valine (V)-267, the serine(S)-268, and the arginine (R)-386. The threonine (T)-97 may besubstituted with alanine (A) or leucine (L), the glutamine (Q)-149 maybe substituted with arginine (R), the proline (R)-166 may be substitutedwith arginine (R), and the proline (P)-351 may be substituted withserine (S). In an example of the exemplary embodiment, in the hexuronateC4-epimerase variant of the present invention, the threonine (T)-272 maybe substituted with aspartic acid (D), valine (V), isoleucine (I),leucine (L), methionine (M), glutamine (Q), or serine (S), the serine(S)-125 may be substituted with aspartic acid (D), the valine (V)-267may be substituted with methionine (M), the serine (S)-268 may besubstituted with cysteine(C) or threonine (T), the arginine (R)-386 maybe substituted with valine (V), or the threonine (T)-97 may besubstituted with alanine (A) or leucine (L), or the glutamine (Q)-149may be substituted with arginine (R), the proline (P)-166 may besubstituted with arginine (R) or the proline (P)-351 may be substitutedwith serine (S).

In an exemplary embodiment, in the hexuronate C4-epimerase variant ofthe present invention, one or more amino acid residues selected from thegroup consisting of the lysine (K)-164, the aspartic acid (D)-168, andthe glutamic acid (E)-175 from the N-terminal of the hexuronateC4-epimerase consisting of the amino acid sequence of SEQ ID NO: 1 mayadditionally be mutated in addition to the threonine (T)-272, serine(S)-125, and valine (V)-267 and/or serine (S)-268 amino acid residues.The lysine (K)-164 may be substituted with methionine (M), the asparticacid (D)-168 may be substituted with glutamic acid (E), and the glutamicacid (E)-175 may be substituted with glycine (G).

According to another exemplary embodiment of the present invention,there is provided a hexuronate C4-epimerase variant in which a leucine(L)-77 amino acid residue from N-terminal of the hexuronate C4-epimeraseconsisting of an amino acid sequence of SEQ ID NO: 1 is mutated. Theleucine (L)-77 may be substituted with proline (P) or arginine (R).

In an exemplary embodiment, in the hexuronate C4-epimerase variant ofthe present invention, the serine (S)-125 amino acid residue from theN-terminal of the hexuronate C4-epimerase consisting of the amino acidsequence of SEQ ID NO: 1 may additionally be mutated in addition to theleucine (L)-77 amino acid residue. The serine (S)-125 may be substitutedwith cysteine (C), tyrosine (Y), glutamine (Q), glutamic acid (E),threonine (T), asparagine (N), or aspartic acid (D).

In an exemplary embodiment, in the hexuronate C4-epimerase variant ofthe present invention, the alanine (A)-158, the proline (P)-351 or acombination of the amino acid residues from the N-terminal of thehexuronate C4-epimerase consisting of the amino acid sequence of SEQ IDNO: 1 may additionally be mutated in addition to the leucine (L)-77 andserine (S)-125 amino acid residues. The alanine (A)-158 may besubstituted with threonine (T), and the proline (P)-351 may besubstituted with serine (S).

In the hexuronate C4-epimerase variant of the present invention, one ormore amino acid residues selected from the group consisting of thehistidine (H)-9, the glutamic acid (E)-60, and the valine (V)-415 fromthe N-terminal of the hexuronate C4-epimerase consisting of the aminoacid sequence of SEQ ID NO: 1 may additionally be mutated in addition tothe leucine (L)-77, serine (S)-125, and alanine (A)-158 amino acidresidues. Specifically, in the hexuronate C4-epimerase variant of thepresent invention, the leucine (L)-77, the serine (S)-125, the alanine(A)-158, the histidine (H)-9, the glutamic acid (E)-60, and valine(V)-415 amino acid residues may be mutated. The histidine (H)-9 may besubstituted with tyrosine (Y), the glutamic acid (E)-60 may be asparticacid (D), and the valine (V)-415 may be substituted with glutamic acid(E).

According to another exemplary embodiment of the present invention,there is provided a hexuronate C4-epimerase variant in which an alanine(A)-158 amino acid residue from N-terminal of the hexuronateC4-epimerase consisting of an amino acid sequence of SEQ ID NO: 1 ismutated. The alanine (A)-158 may be substituted with threonine (T).

In an exemplary embodiment, in the hexuronate C4-epimerase variant ofthe present invention, the serine (S)-125 amino acid residue from theN-terminal of the hexuronate C4-epimerase consisting of the amino acidsequence of SEQ ID NO: 1 may additionally be mutated in addition to thealanine (A)-158 amino acid residue. The serine (S)-125 may besubstituted with cysteine (C), tyrosine (Y), glutamine (Q), glutamicacid (E), threonine (T), asparagine (N), or aspartic acid (D). In anexample, in the amino acid sequence of SEQ ID NO: 1, the alanine (A)-158may be substituted with threonine (T), and the serine (S)-125 may besubstituted with cysteine (C), tyrosine (Y), glutamine (Q), glutamicacid (E), threonine (T), asparagine (N), or aspartic acid (D). In anexemplary embodiment, in the hexuronate C4-epimerase variant of thepresent invention, one or more amino acid residues selected from thegroup consisting of the glutamine (Q)-149, and the valine (V)-267 andthe proline (P)-351 from the N-terminal of the hexuronate C4-epimeraseconsisting of the amino acid sequence of SEQ ID NO: 1 may additionallybe mutated in addition to the alanine (A)-158 and serine (S)-125 aminoacid residues. The glutamine (Q)-149 may be substituted with arginine(R), the valine (V)-267 may be substituted with methionine (M), and theproline (P)-351 may be substituted with serine (S). Accordingly, theremay be provided variants in which the alanine (A)-158 amino acid residueis substituted with threonine (T), the serine (S)-125 is substitutedwith aspartic acid (D), and the glutamine (Q)-149 is additionallysubstituted with arginine (R) or the valine (V)-267 is substituted withmethionine (M) or the proline (P)-351 is substituted with serine (S).

According to another exemplary embodiment of the present invention,there is provided a hexuronate C4-epimerase variant in which a proline(P)-351 amino acid residue from N-terminal of the hexuronateC4-epimerase consisting of an amino acid sequence of SEQ ID NO: 1 ismutated. The proline (P)-351 may be substituted with serine (S).

In an exemplary embodiment, in the hexuronate C4-epimerase variant ofthe present invention, the serine (5)-125 amino acid residue from theN-terminal of the hexuronate C4-epimerase consisting of the amino acidsequence of SEQ ID NO: 1 may additionally be mutated in addition to theproline (P)-351 amino acid residue. The serine (S)-125 may besubstituted with cysteine (C), tyrosine (Y), glutamine (Q), glutamicacid (E), threonine (T), asparagine (N), or aspartic acid (D).Accordingly, there may be provided a variant in which the proline(P)-351 is substituted with serine (S) and the serine (S)-125 issubstituted with cysteine (C), tyrosine (Y), glutamine (Q), glutamicacid (E), threonine (T), asparagine (N), or aspartic acid (D).

In an exemplary embodiment, in the hexuronate C4-epimerase variant ofthe present invention, the valine (V)-267 amino acid residue mayadditionally be mutated in addition to the proline (P)-351 and serine(S)-125 amino acid residues. The valine (V)-267 may be substituted withmethionine (M). In the hexuronate C4-epimerase variant, one Or moreamino acid residues selected from the group consisting of the tyrosine(Y)-21, the valine (V)-62, the glutamine (Q)-149 and the leucine (L)-316may additionally be mutated. The tyrosine (Y)-21 may be substituted withphenylalanine (F), the valine (V)-62 may be substituted with isoleucine(I), the glutamine (Q)-149 may be substituted with arginine (R), and theleucine (L)-316 may be substituted with phenylalanine (F). In an exampleof the exemplary embodiment, the variant may be a variant in which theproline (P) −351 is substituted with serine (S), the serine (S) −125 issubstituted with aspartic acid (D), the valine (V)-267 is substitutedwith methionine (M), the tyrosine (Y)-21 is substituted withphenylalanine (F), the valine (V)-62 is substituted with isoleucine (I),and the glutamine (Q)-149 is substituted with arginine (R), and theleucine (L)-316 is substituted with phenylalanine (F).

Still another object of the present invention is to provide a hexuronateC4-epimerase variant in which a serine (S)-125 amino acid residue, alysine (K)-164 amino acid residue, an aspartic acid (D)-168 amino acidresidue, and a glutamic acid (E)-175 amino acid residue from N-terminalof a hexuronate C4-epimerase consisting of an amino acid sequence of SEQID NO: 1 are mutated. The serine (S)-125 may be substituted withcysteine (C), tyrosine (Y), glutamine (Q), glutamic acid (E), threonine(T), asparagine (N), or aspartic acid (D), the lysine (K)-164 may besubstituted with methionine (M), the aspartic acid (D)-168 may besubstituted with glutamic acid (E), and the glutamic acid (E)-175 may besubstituted with glycine (G).

In an exemplary embodiment, in the hexuronate C4-epimerase variant inwhich the serine (S)-125, lysine (K)-164, aspartic acid (D)-168 andglutamic acid (E)-175 amino acid residues are mutated, one or more aminoacid residues selected from the group consisting of leucine (L)-140,arginine (R)-386, serine (S)-268 and asparagine (N)-297 from theN-terminal of the hexuronate C4-epimerase consisting of the amino acidsequence of SEQ ID NO: 1 may additionally be mutated. The leucine(L)-140 may be substituted with proline (P), the arginine (R)-386 may besubstituted with proline (P) or valine (V), the serine (S)-268 may besubstituted with cysteine (C) or threonine (T), and the asparagine(N)-297 may be substituted with lysine (K). In an example of theexemplary embodiment, the variant may be a variant in which the serine(S)-125 is substituted with aspartic acid (D), the lysine (K)-164 issubstituted with methionine (M), the aspartic acid (D)-168 issubstituted with glutamic acid (E), the glutamic acid (E)-175 issubstituted with glycine (G), the leucine (L)-140 is substituted withproline (P), and the arginine (R)-386 is substituted with proline (P).The hexuronate C4-epimerase variant of the present invention accordingto an example of the exemplary embodiment may be a variant in which theserine (S)-125 is substituted with aspartic acid (D), the lysine (K)-164is substituted with methionine (M), the aspartic acid (D)-168 issubstituted with glutamic acid (E), the glutamic acid (E)-175 issubstituted with glycine (G), the serine (S)-268 is substituted withthreonine (T), and the asparagine (N)-297 is substituted with lysine(K).

According to still another exemplary embodiment of the presentinvention, there is provided a hexuronate C4-epimerase variant in whichserine (S)-125, glutamine (Q)-149, and valine (V)-267 amino acidresidues from N-terminal of a hexuronate C4-epimerase consisting of anamino acid sequence of SEQ ID NO: 1 are mutated. The serine (S)-125 maybe substituted with cysteine (C), tyrosine (Y), glutamine (Q), glutamicacid (E), threonine (T), asparagine (N), or aspartic acid (D), theglutamine (Q)-149 may be substituted with arginine (R), and the valine(V)-267 may be substituted with methionine (M).

According to an exemplary embodiment of the present invention, thehexuronate C4-epimerase variant of the present invention may include apolypeptide moiety having at least 50% genetic identity as compared to ahexuronate C4-epimerase variant consisting of an amino acid sequence(for example, an M125 variant in Tables 2 to 10) capable of beingderived from mutated amino acid residue positions and substituted aminoacid residues disclosed in Tables 2 to 9 in the amino acid sequence (SEQID NO: 1) of the wild-type hexuronate C4-epimerase, or a variant havingthe amino acid sequence, and according to an exemplary embodiment of thepresent invention, the hexuronate C4-epimerase variant of the presentinvention may include a polypeptide moiety having at least 60%, 70%,75%, 80%, 85%, 90%, 95%, or 97% to 99% identity.

As used herein, the term “identity” refers to a percentage of identitybetween two polypeptide moieties. The correspondence between sequencesfrom one moiety to another moiety may be determined by known techniques.For example, the identity may be determined by directly aligningsequence information between the two polypeptide molecules using acomputer program in which the sequence information is aligned andreadily available. Further, the identity may be determined byhybridization of the polynucleotide under a condition in which a stabledouble strand is formed between homologous regions, followed bydegradation by a single-strand-specific nuclease to determine a size ofthe degraded fragment.

As used herein, all grammatical forms or spelling-modified forms of theterm “identical” include superfamily derived proteins (e.g.,immunoglobulin superfamily) and homologous proteins derived from otherspecies (e.g., myosin light chain, etc.,), and refer to a relationshipbetween proteins having a “common evolutionary origin”. The proteins(and coding genes thereof) have sequence identities that are reflectedby a high degree of sequence similarity. However, the term “identical”in the general use and in the present invention refers to sequencesimilarity and does not mean a common evolutionary origin when referredto by an adjective such as “very high”.

As used herein, the term “sequence similarity” refers to the degree ofidentity or correspondence between base sequences or amino acidsequences of a protein that may or may not share the common evolutionaryorigin. In an exemplary embodiment, when two amino acid sequences haveat least 21% (at least about 50% in an embodiment, and at least 75%,90%, 95%, 96%, 97% or 99% in another embodiment) of the polypeptidematch with respect to a predetermined length of the amino acid sequence,they are “substantially identical” or “substantially similar”. Thesubstantially identical sequence may be identified by using standardsoftware used in a data bank, or for example, by comparing sequences bySouthern hybridization experiment under stringent conditions defined fora particular system. Appropriate hybridization conditions to be definedare within the range in the art (e.g., Sambrook et al., 1989, seeinfra).

The hexuronate C4-epimerase variants described herein have improvedC4-epimerase unit activity in which D-fructose is converted intoD-tagatose by epimerizing D-fructose at carbon number 4, therebyefficiently producing D-tagatose from D-fructose.

The hexuronate C4-epimerase variant of the present invention may bederived from hexuronate C4-epimerase of thermophilic microorganismsincluded in the genus Rhodothermus, the genus of Thermoanaerobacter, thegenus of Thermotoga, or the genus of Dictyoglomus. Specifically, thevariant may be derived from the hexuronate C4-epimerase of the genusThermotoga microorganisms, and more specifically, may be derived fromthe hexuronate C4-epimerase of Thermotoga neapolitana or Thermotogamaritima.

The hexuronate C4-epimerase of the present invention may perform astable reaction under the extreme reaction (high temperature, etc.)conditions while having the same function as the enzyme produced bymesophilic microorganisms (mesophile), and may have a number ofadvantages such as prevention of contamination against the mesophilicmicroorganisms, increase of solubility of materials having lowsolubility of substrate, and increase of reaction rate, etc. Thus, it isadvantageous in that it is possible to overcome industrial disadvantagesusing the mesophilic enzyme.

The hexuronate C4-epimerase variants of the present invention may beobtained by transforming a strain such as E. coli, or the like, with DNAexpressing the hexuronate C4-epimerase variant of the present invention,culturing the transformed strain to obtain a culture, crushing theculture, followed by purifying through a column, etc. Examples of thestrain for the transformation include Escherichia coli, Corynebacteriumglutamicum, Aspergillus oryzae, Bacillus subtilis, etc.

According to another exemplary embodiment of the present invention, theprevent invention provides a nucleic acid encoding a hexuronateC4-epimerase variant as described in the present invention, atransformant including the nucleic acid, or a composition for productionof D-tagatose comprising a microorganism expressing the hexuronateC4-epimerase variant described in the present invention or a culture ofthe microorganism or the hexuronate C4-epimerase variant described inthe present invention.

Another exemplary embodiment is directed to an expression vectorincluding the nucleic acid encoding a hexuronate C4-epimerase variantdescribed in the present invention. The term “vector” in the presentinvention refers to any mediator for cloning and/or transferring ofbases into an organism, such as a host cell. The vector may be areplicon that is able to bring the replication of combined fragments inwhich different DNA fragments are combined. Here, the term “replicon”refers to any genetic unit (e.g., plasmid, phage, cosmid, chromosome,virus) which functions as a self-unit of DNA replication in vivo, i.e.,which is able to be replicated by self-regulation. The term “vector”includes viral and nonviral mediators for introducing the bases into theorganism, e.g., a host cell, in vitro, ex vivo or in vivo. The term“vector” may also include mini-spherical DNA.

The term “nucleic acid” as used herein means that it encompasses DNA orRNA molecules, wherein nucleotides which are basic constituent units inthe nucleic acid may include not only natural nucleotides but alsoanalogues in which sugar or base sites are modified (see Scheit,Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman,Chemical Reviews, 90:543-584(1990)).

The term “transformation” as used herein means that the nucleic acidfragment migrates into the genome of a host organism to causegenetically stable transition, and the term “transformant” refers to anorganism in which the genetically stable transition is caused by themigration of the nucleic acid in the genome thereof. The transformantmay be, for example, a prokaryotic cell or a eukaryotic cell.Specifically, the transformant may be Enterobacteriaceae microorganismor coryneform microorganisms, etc., more specifically, the genusEscherichia microorganism, the genus Serratia microorganism, etc., andthe most specifically, E. coli.

A method for transformation into an organism includes any method forintroducing the nucleic acid into an organism and may be performed byappropriately selecting appropriate standard techniques as known in theart. As an example, the method includes electroporation, calciumphosphate co-precipitation, retroviral infection, microinjection,DEAE-dextran, cationic liposome method, etc., but is not limitedthereto.

The composition for production of D-tagatose comprising a hexuronateC4-epimerase variant may further include any suitable excipientconventionally used in the composition for production of D-tagatose. Theexcipient may be, for example, but is not limited to, preservatives,wetting agents, dispersing agents, suspending agents, buffers,stabilizing agents, isotonic agents, or the like. The hexuronateC4-epimerase variant in the composition may be included in the range of0.1 wt % to 70 wt % based on the solid weight of the composition.

According to still another exemplary embodiment of the presentinvention, the prevent invention provides a method for production ofD-tagatose comprising: contacting the hexuronate C4-epimerase variantdescribed in the present invention, the transformant described in thepresent invention, or the composition for production of tagatosedescribed in the present invention with D-fructose to epimerize theD-fructose.

Hereinafter, the method for production of D-tagatose according to anexemplary embodiment of the present invention is described.

The method may include contacting the hexuronate C4-epimerase variant ofthe present invention, a microorganism expressing the variant or aculture of the microorganism or a composition for production ofD-tagatose comprising the same with D-fructose. Thus, it is possible toepimerize D-fructose at carbon number 4.

Monosaccharides may be generally classified into aldohexose andketohexose. D-fructose as a raw material in the present invention is anexample of ketohexose, and the D-fructose may be used to produceD-tagatose.

The D-fructose may be produced by hydrolysis of sugar, or may beproduced by isomerizing glucose. As a result, it is possible to producetagatose at a high yield by using a universal and inexpensive rawmaterial such as fructose, sugar and glucose, thereby enabling massproduction of tagatose.

A step for epimerization of D-fructose of the present invention may beperformed at a pH from 5 to 9, at a pH from 6 to 9, at a pH from 7 to 9,or at a pH from 7.5 to 8.5. The step for epimerization of D-fructose ofthe present invention may be performed at 50° C. to 85° C., 50° C. to75° C. or 50° C. to 70° C. When treating the variant enzyme of thepresent invention under the above-described pH or temperatureconditions, a reaction is able to proceed at a relatively hightemperature, and thus, it is possible to minimize microbialcontamination during the production process, to increase solubility offructose used as a substrate, and to maximize a reaction rate and aconversion rate of the enzyme.

Further, the D-fructose of the present invention may have aconcentration of 10 to 50% (w/v). According to an exemplary embodiment,the concentration may be 20 to 50% (w/v), 20 to 40% (w/v), 20 to 30%(w/v). The variant enzyme of the present invention is capable ofproducing D-tagatose from a high concentration of D-fructose, and thusis economically and efficiently capable of producing D-tagatose.

The step for epimerization of the D-fructose of the present inventionmay be performed in the presence of a metal salt. In an exemplaryembodiment, a metal in the metal salt of the present invention may be atleast one metal selected from the group consisting of Ni, Co, Mn, andZn. Specifically, the metal salt of the present invention may be atleast one selected from the group consisting of NiSO₄, NiCl₂, CoCl₂,MnCl₂, and ZnSO₄. Since the step for epimerization of D-fructose of thepresent invention is performed in the presence of the metal salt, aneffect of improving the conversion activity is able to be obtained.

According to an exemplary embodiment of the present invention, theproduction method of the present invention may further include, beforethe contacting step of the present invention, hydrolyzing sugar toobtain D-fructose. The enzyme used for the hydrolysis may include atleast one selected from the group consisting of β-D-fructosidaseincluding β-fructofuranosidase, invertase, and saccharase, etc.;sucrase, α-glucosidase and α-D-glucohydrolase, but is not limitedthereto.

According to an exemplary embodiment of the present invention, theproduction method of the present invention may further include, beforethe contacting step of the present invention, isomerizing glucose toobtain D-fructose. The isomerase may be glucose isomerase orphosphogluco isomerase, but is not limited thereto.

According to an exemplary embodiment of the present invention, theproduction method of the present invention may further include, afterthe contacting step of the present invention, obtaining an epimerizationreaction product including D-tagatose.

According to an exemplary embodiment of the present invention, theproduction method of the present invention may further include, afterthe obtaining step of the epimerization reaction product of the presentinvention, purifying the obtained epimerization reaction productincluding D-tagatose.

According to an exemplary embodiment of the present invention, theproduction method of the present invention may further include, afterthe purifying step of the obtained epimerization reaction product of thepresent invention, crystallizing the purified epimerization reactionproduct including D-tagatose.

A method for purification of the epimerization reaction product is notparticularly limited, and may be a method commonly used in the technicalfield of the present invention. Non-limiting examples thereof mayinclude chromatography, fractional crystallization, ion purification,etc. The purification method may be performed only by one method, or byperforming two or more methods together. For example, the epimerizationreaction product may be purified through chromatography, and separationof the sugar by the chromatography may be performed by utilizing adifference in weak binding force between the sugar to be separated andthe metal ion attached to an ion resin.

In addition, the present invention may further include performingdecolorization, desalination or both of decolorization and desalinationbefore or after the purification step of the present invention. Byperforming the decolorization and/or desalination, it is possible toobtain a more purified epimerization reaction product withoutimpurities.

The purified epimerization reaction product may be concentrated toobtain a pure tagatose solution through an SMB chromatography process,followed by crystallization.

According to an exemplary embodiment of the present invention, theproduction method of the present invention may further include, beforethe crystallizing step of the present invention, concentrating theseparated pure tagatose solution. The concentrating step may beperformed to have a concentration of the epimerization reaction productincluding the purified D-tagatose about 2.5 to 3 times, and thecrystallization may be performed more efficiently through theconcentration step.

The method used in the crystallizing step of the present invention isnot particularly limited, and may be a commonly used crystallizationmethod. For example, a crystallization method using a coolingcrystallization method may be used. Through the crystallizing step, itis possible to obtain finally purified D-tagatose at a high yield.

According to an exemplary embodiment of the present invention, theproduction method of the present invention may further include, afterthe purifying step of the present invention, reusing unreactedD-fructose in the contacting step of the present invention, or after thecrystallizing step of the present invention, reusing a mother liquorfrom which a crystal is separated in the purifying step, or performingboth steps. The reusing step is economically advantageous sinceD-tagatose may be obtained at a higher yield, and an amount ofD-fructose to be discarded may be reduced.

The term “carbon number n” as used herein refers to a carbon positiondetermined in accordance with carbon numbering prescribed in IUPACnomenclature, and may be expressed as Cn. Here, n is an integer of 1 ormore. For instance, “epimerization at carbon number 4” is represented by“C4-epimerization”.

The amino acid residue (X) at the n-th position from the N-terminal ofthe hexuronate C4-epimerase consisting of the amino acid sequence of SEQID NO: 1 may be abbreviated as n X in the present invention.

In addition, it is possible to consider amino acids substitutable at theamino acid residues at corresponding positions mentioned in other partsof the present invention, unless otherwise stated herein with respect toamino acids which are substituted in the amino acid residues to bemutated in the present invention.

Amino acids in the present invention may be denoted by the followingabbreviations or amino acid names.

TABLE 1 Amino acid type Abbreviation Alanine A Arginine R Asparagine NAspartic acid D Cysteine C Glutamic acid E Glutamine Q Glycine GHistidine H Isoleucine I Leucine L Lysine K Methionine M Phenylalanine FProline P Serine S Threonine T Tryptophan W Tyrosine Y Valine V

In addition, the disclosures of Korean Patent Laid-Open Publication No.10-2014-0143109 are incorporated herein by reference.

Advantageous Effects

The present invention provides a hexuronate C4-epimerase variant havingimproved activity of converting D-fructose into D-tagatose byepimerizing D-fructose at carbon number 4, thereby efficiently enablingmass-production of D-tagatose using D-fructose which is a universal rawmaterial, and thus the production cost may be reduced to provideeconomic advantages.

BEST MODE

Hereinafter, the present invention is described in more detail withreference to the following Examples. However, the following Examples aremerely examples of the present invention, and the contents of thepresent invention should not be construed as being limited thereto.

EXAMPLE Example 1. Improved Target Site Design and Analysis

Amino acids predicted to be functionally important were firstly selectedbased on analysis of the tertiary structure model of the active site ofthe ortholog (a homologous gene predicted to have the same function indifferent microbial species) which has identity with an amino acid of ahexuronate C4-epimerase derived from Thermotoga neapolitana (hereinafterreferred to as wild-type). Then, based on analysis results of thedocking model between D-fructose and the refined active site structureafter the alanine scanning mutagenesis, a modified target site wasdesigned for improvement of the unit activity of the conversion reactionof D-fructose by C4-epimerization. The details thereof are described asfollows.

1-1. Ortholog Analysis

The homologous genes (ortholog) having identity with the wild-type aminoacid sequence (SEQ ID NO: 1) [about 60 homologous genes with 80%sequence coverage and 50% or more homology] were screened using GenBankgene database. Through multiple sequence alignment analysis among aminoacid sequences of the selected homologous genes, conserved amino acidresidues predicted to be functionally important in the wild-type aminoacid sequence were identified.

1-2. Analysis of Enzyme Tertiary Structure Model

There was no protein structure that appears to have 30% or more aminoacid sequence identity with the homologous genes of the wild-type inProtein Data Bank database, and thus it was expected that accuracy inthe prediction of the tertiary structure model of the wild-type by ahomology modeling method would be low. Accordingly, the active sitesamong the tertiary structure models obtained from various modelingservers (RaptorX, Robetta, ModWeb, M4T, HHpred, PHYRE2, ITASSER andSWISS-MODEL) were compared and analyzed to obtain information about thestructure sites that were predicted as the same.

1-3. Alanine Scanning Mutagenesis and Docking Binding Analysis

The amino acids that were selected based on the amino acid sequenceanalysis and the analysis of the tertiary structural model of the activesite among the homologous genes as described above were substituted andmutated with alanine, and these recombinant mutation enzymes wereproduced in Escherichia coli. Then, characteristics of each mutationsite were analyzed. Amino acids predicted to be functionally importantwere selected through the docking simulation between D-fructose and therefined active site structure after the alanine scanning mutagenesis wasanalyzed. Then, the modified target site was designed for theimprovement of the unit activity of the conversion reaction ofD-fructose by C4-epimerization. The amino acid sites of which activityis completely lost through the alanine scanning mutagenesis [assumingcatalytic metal ion binding residues and deprotonation/protonationinvolved catalytic residues] were excluded from the target site foractivity improvement.

Example 2. Production of Mutation Enzyme and Selection ofActivity-Modified Mutation Enzyme

Single-site saturation mutagenesis libraries of 54 target sites designedin Example 1 (amino acid residues at position Nos: 9, 21, 60, 62, 68,77, 91, 97, 125, 126, 140, 141, 145, 149, 157, 158, 160, 163, 164, 166,167, 168, 175, 176, 177, 185, 202, 218, 221, 231, 241, 242, 267, 268,272, 276, 284, 295, 297, 302, 306, 316, 337, 351, 361, 366, 386, 388,402, 403, 415, 429, 440, and 441 from the N-terminal of the wild-typehexuronate C4-epimerase) were constructed, and mutation sites of whichthe unit activity was improved and amino acids were screened. Themultiple mutation enzyme was made by integrating the information of thescreened modified sites to develop a mutation enzyme having improvedunit activity of the conversion reaction of D-fructose byC4-epimerization.

2-1. Saturation Mutagenesis

The recombinant expression vector constructed for expression ofwild-type enzyme gene, wild-type Escherichia coli BL21 (DE3) (whichexpresses the recombinant enzyme in which the wild-type was introducedinto the Ndel and Xhol restriction enzyme sites of pET21a and 6×His-tagis bound at the C-terminal of the wild-type) was used as a template forsaturation mutagenesis for producing a variant library. InversePCR-based saturation mutagenesis was used in consideration of diversityof mutation distribution and yield of variants (2014. Anal. Biochem.449: 90-98), NDT, VMA, ATG and TGG mixed primers in which terminationcodon was excluded and rare codons of E. coli were minimized in order tominimize the screening scale of the constructed variant library (i.e.,to minimize the number of codons introduced during saturationmutagenesis) were designed and used (2012. Biotechniques 52:149-158).Specifically, a mixed primer including 15 bp for the front base, 3 bp(NDT, VMA, ATG and TGG, respectively) for substituting the displacedsite, and 15 bp for the back base of the respective mutated sites, i.e.,33 bp in total length was constructed and used. The PCR was repeated 30times under conditions of denaturation at 94° C. for 2 minutes,denaturation at 94° C. for 30 seconds, annealing at 60° C. for 30seconds, extension at 72° C. for 10 minutes, and extension at 72° C. for60 minutes. After constructing the saturation mutagenesis libraries foreach mutation site, variants for each library were randomly selected(<mutation 11), and base sequences were analyzed to evaluate amino acidmutation distribution. Based on the analysis results, the screeningscale of 90% or more of the sequence coverage for each library wasdetermined (2003. Nucleic Acids Res. 15; 31:e30).

2-2. Screening of Activity-Modified Mutation Enzyme and Construction ofMultiple Mutation Enzyme

A chromogenic assay was used to specifically quantify D-fructose inorder to rapidly screen large quantities of activity-modified mutationenzymes in the produced saturation mutagenesis libraries. Specifically,a 70% folin-ciocalteu reagent (SIGMA-ALDRICH) and a substrate reactionsolution were mixed at a ratio of 15:1 and reacted at 80° C. for 5minutes. The OD values measured at 900 nm were compared and analyzed.

54 variants in the mutation site with increased activity (D-tagatoseproduction by conversion of D-fructose) as compared to the relativeactivity of the wild-type enzyme (SEQ ID NO: 1) were firstly selected.The base sequences of the corresponding genes were analyzed and theamino acid mutation information was analyzed (Tables 2 to 10).

The firstly selected mutation enzymes were reacted with D-fructose usinga purified enzyme solution (His-tag affinity chromatography), and thereaction products were used to finally select 236 variants with theincreased activity in producing D-tagatose by conversion from D-fructoseas compared to the wild-type enzyme by using HPLC (column Shodex SUGARSP-G, column analysis temperature of 80° C., mobile phase H₂O, flow rateof 0.6 ml/min, Refractive Index Detector).

Example 3. Comparative Evaluation of Activity-Modified Mutation EnzymeCharacteristics

In order to evaluate the relative activity of the D-fructoseC4-epimerization on the mutation enzyme for a single site with improvedunit activity and on the mutation enzyme for a multiple site incombination thereof, each enzyme was expressed in E. coli BL21 (DE3) bya conventional method (see Sambrook et al. 1989) and purified (byHis-tag affinity chromatography). Then, in the presence of NiSO₄, eachenzyme at a concentration of 10 units/ml was added to 25% (w/v)D-fructose substrate and reacted at pH 8.0 [50 mM potassium phosphatebuffer] and at 65° C. for 2 hours, and the relative activity ofD-fructose C4-epimerization as compared to the wild-type recombinase(wild-type, SEQ ID NO: 1) derived from Thermotoga neapolitana wasmeasured.

TABLE 2 name/ mutation Number of relative position 77 125 158 185 272403 variants activity WT — 100 M1 C 1 193 M2 Y 1 116 M3 Q 1 165 M4 E 1202 M5 T 1 211 M6 N 1 131 M7 D 1 303 M8 K 1 114 M9 R 1 114 M10 H 1 118M11 Q 1 107 M12 A 1 102 M13 F 1 104 M14 E 1 120 M15 D 1 121 M16 Q 1 116M17 S 1 133 M18 V 1 117 M19 R 1 105 M20 K 1 117 M21 S 1 114 M22 T 1 130M23 Q 1 119 M24 F 1 110 M25 V 1 117 M26 I 1 128 M27 A 1 119 M28 P D 2479 M29 P D 2 487 M30 R D 2 426 M31 D T 2 494 M32 D K 2 543 M33 D R 2430 M34 D H 2 493 M35 D Q 2 584 M36 D A 2 447

TABLE 3 name/ mutation Number of relative position 77 125 149 158 185267 268 272 351 403 variants activity M37 D G 2 481 M38 D S 2 355 M39 DP 2 199 M40 D D 2 343 M41 D H 2 259 M42 D Q 2 347 M43 D N 2 222 M44 D K2 351 M45 D H 2 343 M46 D Y 2 305 M47 D S 2 421 M48 D S 2 377 M49 D F 2380 M50 D T 2 431 M51 D Q 2 371 M52 D V 2 372 M53 R D T 3 572 M54 P D T3 452 M55 P D S 3 473 M56 D R T 3 557 M57 D R M 3 594 M58 D T M 3 608M59 D T S 3 605 M60 D T S 3 480 M61 D Q C 3 422 M62 D Q C 3 422 M63 D KD 3 638 M64 D K V 3 402 M65 D K I 3 515 M66 D K L 3 506 M67 D K M 3 540M68 D K Q 3 628 M69 D K T 3 790 M70 D Q T 3 746 M71 D M S 3 613 M72 D DF 3 281 M73 D D S 3 274 M74 D D V 3 178 M75 D D I 3 314

TABLE 4 name/ mutation position 9 21 60 62 68 77 97 125 140 149 158 164166 168 175 185 231 M76 R D T M77 D Q M78 D Q M79 R D T M80 D Q M81 D QM82 D R M83 D M84 D M85 Y D P D T M86 G D Q M87 L D M88 D P M E G M89 DR M90 D M E G M91 D R M92 D Q M93 D M94 D Q M95 F I D R M96 F I D R M97F I D R M98 F I D R name/ mutation Number of relative position 267 268272 297 306 316 351 386 403 415 variants activity M76 T 4 441 M77 H M 4495 M78 D M 4 548 M79 V T 5 437 M80 C D M 5 526 M81 M T D 5 451 M82 M TD 5 510 M83 M T D V 5 555 M84 M S V T 5 445 M85 E 6 427 M86 M M T 6 489M87 M T D V 6 695 M88 V 6 564 M89 M T D V 6 496 M90 T K 6 498 M91 M T DV 6 592 M92 M T D T 6 691 M93 M T D S V 6 553 M94 M C D M 6 588 M95 M FS 7 540 M96 M F S 7 454 M97 M F S 7 498 M98 M F S 7 500

TABLE 5 name/ mutation Number of relative position 91 125 141 164 168175 176 185 267 268 272 306 403 variants activity M99 W D Q M T D T 7478 M100 I D Q M T D T 7 560 M101 N D Q M T D T 7 486 M102 D F Q M T D T7 496 M103 D M E G M C D 7 437 M104 D H Q M T D I 7 610 M105 D F Q M T DI 7 539 M106 D Y Q M T D I 7 662 M107 D Q M T D M Q 7 822 M108 D Q M T DM I 7 1011 M109 D Q M T D M L 7 728 M110 D Q M T D M A 7 749 M111 D Q MT D M P 7 728 M112 D Q M T D M V 7 1023 M113 D Q M T D M W 7 682 M114 DQ M T D M R 7 607 M115 D Q M T D M H 7 948 M116 D Q M T D M F 7 956 M117D Q M T D M K 7 536 M118 D Q M T D M N 7 932 M119 D Q M T D M E 7 400M120 D Q M T D M D 7 476 M121 D Q M T D M C 7 457 M122 D Q M T D M T 7690

TABLE 6 name/ mutation Number of relative position 125 126 164 166 168175 185 231 267 268 272 297 306 386 388 403 variants activity M123 D Q MT D M V 7 326 M124 D Q M T D V T 7 693 M125 D Q M T M V T 7 822 M126 D QM D M V T 7 558 M127 D Q T D M V T 7 655 M128 D M T D M V T 7 597 M129 QM T D M V T 7 589 M130 D G Q M T D V T 8 521 M131 D M E G T K V T 8 445M132 D R Q M T D M T 8 697 M133 D Q R M T D M T 8 640 M134 D Q M T G M VM 8 487 M135 D Q M T D M V M 8 786 M136 D Q M T D M V G 8 808 M137 D Q MT D H V T 8 440 M138 D Q M T D V V T 8 649 M139 D Q M T D F V T 8 740M140 D Q M T D M V I 8 1006 M141 D Q M T V M V I 8 699 M142 D Q M T A MV I 8 540 M143 D Q M C D M V T 8 495 M144 D Q M T E M V I 8 931 M145 D QM T D M V G 8 557 M146 D Q M T D M V D 8 625

TABLE 7 name/ mutation position 60 97 125 126 145 163 164 166 185 202221 231 241 242 M147 D Q M148 D M149 D D T F F M150 A D Q M151 L D QM152 D F Q M153 D L Q M154 D P Q M155 D I Q M156 D T Q M157 D A Q M158 DG Q M159 D R Q M160 D A Q M161 D A Q M162 D Q Q M163 D M Q M164 D R QM165 D Q R M166 D Q N M167 D Q T M168 D Q S M169 D Q M170 D Q name/mutation Number of relative position 267 268 272 276 284 306 386 403 415variants activity M147 M T D M V N 8 408 M148 M C D A M T E 8 418 M149 MT P T 9 643 M150 M T D M V T 9 672 M151 M T D M V T 9 695 M152 M T D M VT 9 661 M153 M T D M V T 9 656 M154 M T D M V T 9 636 M155 M T D M V T 9667 M156 M T D M V T 9 670 M157 M T D M V T 9 518 M158 M T D M V I 9 682M159 M T D M V I 9 553 M160 M T D M V T 9 553 M161 M T D M V T 9 664M162 M T D M V T 9 597 M163 M T D M V T 9 634 M164 M T D M V T 9 752M165 M T D M V T 9 733 M166 M T D M V I 9 699 M167 M T D M V I 9 697M168 M T D M V I 9 736 M169 M T D E M V I 9 601 M170 M T D A M V I 9 586

TABLE 8 name/ mutation position 97 125 157 160 163 164 166 167 185 231267 268 272 306 M171 D Q M T D M M172 D Q M T D M M173 D Q M T D M M174D Q M T D M M175 D Q M T D M M176 D Q M T D M M177 D Q M T D M M178 D QM T D M M179 D Q M T D M M180 D Q M T D M M181 D Q M T D M M182 D Q M TD M M183 D Q M T D M M184 L D M Q M T D M M185 L D R Q M T D M M186 L DQ R M T D M M187 D R Q M T D M M188 D L Q M T D M M189 D F Q M T D MM190 D R Q M T D M M191 D Y Q M T D M M192 D M Q R M T D M M193 D A Q MT D M M194 D W Q M T D M name/ mutation Number of relative position 337366 386 402 403 429 440 variants activity M171 T V I 9 1093 M172 Y V I 91093 M173 N V I 9 1489 M174 P V I 9 1408 M175 S V I 9 1180 M176 S V I 9771 M177 G V I 9 367 M178 C V I 9 476 M179 V F I 9 677 M180 V C I 9 668M181 V Y I 9 644 M182 V T P 9 585 M183 V T A 9 764 M184 V T 10 550 M185V T 10 706 M186 V T 10 613 M187 W V I 10 1268 M188 W V I 10 1429 M189 WV I 10 982 M190 W V I 10 565 M191 W V I 10 668 M192 V T 10 617 M193 W VI 10 1803 M194 W V I 10 1854

TABLE 9 name/ mutation Number of relative position 125 167 177 185 218267 268 272 295 306 337 386 403 variants activity M195 D I Q M T D M W VI 10 1678 M196 D K Q M T D M W V I 10 1432 M197 D M Q M T D M W V I 101770 M198 D V Q M T D M W V I 10 1351 M199 D S Q M T D M W V I 10 1951M200 D Y Q M T D M W V I 10 911 M201 D H Q M T D M W V I 10 733 M202 D LQ M T D M W V I 10 1489 M203 D Q I M T D M W V I 10 818 M204 D Q S M T DM W V I 10 1294 M205 D Q L M T D M W V I 10 1348 M206 D Q F M T D M W VI 10 1350 M207 D Q C M T D M W V I 10 1204 M208 D Q M T D C M W V I 101000 M209 D Q M T D R M W V I 10 485 M210 D Q M T D Y M W V I 10 1261

TABLE 10 name/ mutation Number of relative position 125 185 267 268 272302 306 337 361 366 386 403 441 variants activity M211 D Q M T D C M W VI 10 1222 M212 D Q M T D M W K V I 10 966 M213 D Q M T D M W E V I 10630 M214 D Q M T D M W V V I 10 586 M215 D Q M T D M W W V I 10 783 M216D Q M T D M W Y V I 10 781 M217 D Q M T D M W M V I 10 549 M218 D Q M TD M W R V I 10 760 M219 D Q M T D M W Q V I 10 731 M220 D Q M T D M W LV I 10 638 M221 D Q M T D M W R V I 10 879 M222 D Q M T D M W Y V I 101428 M223 D Q M T D M W C V I 10 856 M224 D Q M T D M W L V I 10 589M225 D Q M T D M F S V I 10 1306 M226 D Q M T D M E S V I 10 1246 M227 DQ M T D M S S V I 10 1271 M228 D Q M T D M W S V I 10 1306 M229 D Q M TD M W V I E 10 1160 M230 D Q M T D M W V I W 10 1150 M231 D Q M T D M WV I H 10 1250 M232 D Q M T D M W V I K 10 1270 M233 D Q M T D M W V I A10 1250 M234 D Q M T D M W V I R 10 1220 M235 D Q M T D M W V I S 101449 M236 D Q M T D M W V I F 10 1294

From the above results, it could be confirmed that the C4-epimerasevariants of the present invention had the increased D-fructoseC4-epimerization activity as compared to that of the wild-type enzyme,and in particular, the enzyme variant of M199 was analyzed as havingincreased the unit activity about 20 times, and thus, it could beconfirmed that the activity of producing tagatose of the presentinvention was remarkably increased as compared to the wild-type enzyme.

1. A nucleic acid encoding a hexuronate C4-epimerase variant in whichserine (S) as an amino acid residue at position 185 from the N-terminusof a hexuronate C4-epimerase consisting of the amino acid sequence setforth in SEQ ID NO: 1 is replaced by alanine (A), glycine (G), histidine(H), lysine (K), glutamine (Q) or arginine (R).
 2. The nucleic acidaccording to claim 1, wherein, in the hexuronate C4-epimerase variant,the serine (S) residue at position 125 from the N-terminus of thehexuronate C4-epimerase is further replaced by cysteine (C), tyrosine(Y), glutamine (Q), glutamic acid (E), threonine (T), asparagine (N) oraspartic acid (D).
 3. The nucleic acid according to claim 1, wherein, inthe hexuronate C4-epimerase variant, the serine (S) residue at position268 from the N-terminus of the hexuronate C4-epimerase is furtherreplaced by cysteine (C) or threonine (T).
 4. A transformant comprisingthe nucleic acid according to claim
 1. 5. The transformant according toclaim 4, wherein, in the hexuronate C4-epimerase variant, the serine (S)residue at position 125 from the N-terminus of the hexuronateC4-epimerase is further replaced by cysteine (C), tyrosine (Y),glutamine (Q), glutamic acid (E), threonine (T), asparagine (N) oraspartic acid (D).
 6. The transformant according to claim 4, wherein, inthe hexuronate C4-epimerase variant, the serine (S) residue at position268 from the N-terminus of the hexuronate C4-epimerase is furtherreplaced by cysteine (C) or threonine (T).
 7. A method for D-tagatoseproduction, comprising bringing a hexuronate C4-epimerase variant inwhich serine (S) as an amino acid residue at position 185 from theN-terminus of a hexuronate C4-epimerase consisting of the amino acidsequence set forth in SEQ ID NO: 1 is replaced by alanine (A), glycine(G), histidine (H), lysine (K), glutamine (Q) or arginine (R).
 8. Themethod according to claim 7, wherein, in the hexuronate C4-epimerasevariant, the serine (S) residue at position 125 from the N-terminus ofthe hexuronate C4-epimerase is further replaced by cysteine (C),tyrosine (Y), glutamine (Q), glutamic acid (E), threonine (T),asparagine (N) or aspartic acid (D).
 9. The method according to claim 7,wherein, in the hexuronate C4-epimerase variant, the serine (S) residueat position 268 from the N-terminus of the hexuronate C4-epimerase isfurther replaced by cysteine (C) or threonine (T).